Figure 7

Figure 7 Consequences of CDKL1 variants for ciliary biology.

(A) Expression of CDKL1 variants affects primary cilia formation. EGFP-CDKL1 variants were transiently expressed in RPE-1 cells (green), serum-starved to induce ciliation, and stained for the axoneme of primary cilia with anti–acetylated tubulin (Ac. tub., magenta). Scale bar: 10 μm. n = 5 experiments with more than 100 cells per condition and experiment counted. **P = 0.0083, Kruskal-Wallis test with Dunn’s multiple-comparison post-test. (B) Expression of disease-associated CDKL1 variants increases number of bi-ciliated RPE-1 cells. EGFP-CDKL1 variants were transiently expressed in RPE-1 cells (green) and stained for the axoneme with anti–acetylated tubulin (Ac. tub., green) and for the basal body with anti–γ-tubulin (G.-tub., magenta) antibodies. Scale bar: 5 μm. n = 3 experiments with 211–441 ciliated cells counted. *P = 0.0309, **P = 0.0070, Brown-Forsythe and Welch’s ANOVA test with Dunnett’s T3 multiple-comparison test. (C) CDKL1 variants localize at the basal body (and tip) of the primary cilium and affect cilia length in HEK293T cells. EGFP-CDKL1 variants were transiently expressed in HEK293T cells (green). Cells were stained for the axoneme of primary cilia with anti–acetylated tubulin (red) and for the basal body with anti-pericentrin (cyan) antibodies. Nuclei were stained with DAPI (blue). Shown are representative images of CDKL1-expressing cells with primary cilia; further images are given in Supplemental Figure 11. Scale bars: 10 μm, and 1 μm in close-up images. Quantification of cilium length: n = 3 experiments with ≥20 cilia per condition measured; median (red bars) and individual experiments (circles) are indicated. **P < 0.01, ****P < 0.0001, Brown-Forsythe and Bartlett’s ANOVA test with Tukey’s multiple-comparison test. Quantification of CDKL1 localization: Stacked bar graphs summarize 3 experiments with ≥17 cilia per condition analyzed. ****P < 0.0001, Fisher’s exact test. All images (A–C) were acquired with a confocal microscope.